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Pharmacologic HIV-1 Nef blockade promotes CD8 T cell–mediated elimination of latently HIV-1–infected cells in vitro
Shariq Mujib, … , Thomas E. Smithgall, Mario A. Ostrowski
Shariq Mujib, … , Thomas E. Smithgall, Mario A. Ostrowski
Published September 7, 2017
Citation Information: JCI Insight. 2017;2(17):e93684. https://doi.org/10.1172/jci.insight.93684.
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Research Article AIDS/HIV Immunology

Pharmacologic HIV-1 Nef blockade promotes CD8 T cell–mediated elimination of latently HIV-1–infected cells in vitro

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Abstract

Eradication of the HIV-1 latent reservoir represents the current paradigm to developing a cure for AIDS. HIV-1 has evolved multiple mechanisms to evade CD8 T cell responses, including HIV-1 Nef–mediated downregulation of MHC-I from the surface of infected cells. Nef transcripts and protein are detectable in samples from aviremic donors, suggesting that Nef expression in latently HIV-1–infected CD4 T cells protects them from immune-mediated clearance. Here, we tested 4 small molecule inhibitors of HIV-1 Nef in an in vitro primary CD4 T cell latency model and measured the ability of autologous ex vivo or HIV-1 peptide–expanded CD8 T cells to recognize and kill latently infected cells as a function of inhibitor treatment. Nef inhibition enhanced cytokine secretion by autologous CD8 T cells against latently HIV-1–infected targets in an IFN-γ release assay. Additionally, CD8 T cell–mediated elimination of latently HIV-1–infected cells was significantly enhanced following Nef blockade, measured as a reduction in the frequency of infected cells and Gag protein in cultures following viral outgrowth assays. We demonstrate for the first time to our knowledge that Nef blockade, in combination with HIV-specific CD8 T cell expansion, might be a feasible strategy to target the HIV-1 latent reservoir that should be tested further in vivo.

Authors

Shariq Mujib, Aamir Saiyed, Saleh Fadel, Ardalan Bozorgzad, Nasra Aidarus, Feng Yun Yue, Erika Benko, Colin Kovacs, Lori A. Emert-Sedlak, Thomas E. Smithgall, Mario A. Ostrowski

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Figure 5

Nef blockade resulted in greater elimination of latently HIV-1–infected CD4 T cells.

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Nef blockade resulted in greater elimination of latently HIV-1–infected ...
Resting latently HIV-1–infected cells were treated with Nef inhibitors for 4–6 days. Cells were then cocultured at a 1:1 ratio with autologous HIV-1 peptide–expanded CD8 T cells in medium containing HAART. One day later, an MHC-I blocking antibody, W6/32, was added to the wells to prevent further CD8 T cell–mediated killing, along with IL-15 to induce virus production. Six days later, the frequency of CD4 T cells producing HIV-1 Gag p24 were enumerated via flow cytometry. Shown in A are representative plots of an experiment with CD4 T cells infected with the NL4-3 virus. (B) Paired data between the control and B9-treated latently NL4-3–infected cell cocultures with expanded CD8 T cells are shown, and the summary data with each indicated treatment is shown. (C) Paired results between cocultures of latently NL4-3 ΔNef–infected cells receiving the inhibitor B9 and the DMSO control are shown. (D) Data were normalized to the DMSO control condition and are reported as the Residual Gag+ CD4 T cells, the percentage of HIV-1 Gag p24+ cells as a fraction of the DMSO control. Paired data between control and B9 cocultures and summary data are shown. Medians shown for B and C. Mean with SEM shown for summary data in D. (B–D) NL4-3 WT: n = 15 B9; n = 14 others. NL4-3 ΔNef: n = 10 B9, n = 8 others. All analyses were performed comparing the control (NT) data to the paired Nef inhibitor data. Wilcoxon matched-pair tests were performed to determine statistical significance in B and C. Paired t tests were performed to determine statistical significance for Residual Gag+ cells (%) data in D. Reported P values have been corrected for multiple comparisons using the Benjamini-Hochberg procedure. *P < 0.05, **P < 0.01, ***P < 0.001.

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